The overall goal of this project is to produce human monoclonal antibodies against human tumor associated antigens (TAA). Two highly specific TAAs have been selected: a prostate specific antigen (PSA, 30 kD glycoprotein) and Carcinoembryonic antigen (CEA, 180 kD glycoprotein). Our approach is to utilize a combination of three methods previously independently used to generate human monoclonal antibodies: a) in vitro immunization of human spleen cells, b) immunization and expansion of human lymphocytes in SCID mice (hu-SCID model), and c) cloning and expression of human antibody genes by construction of M-13 combinatorial libraries. To achieve these goals, we have divided our project into 4 specific aims: I) In vitro immunization to generate B-cells primed against TAAs, II) immunization of SCID mice to further expand and select high affinity antibody producing human B-cell populations, III) construction of combinatorial libraries to clone and expand human antibody genes, and IV) antibody production and biological testing to obtain preclinical data on therapeutic human antibodies for passive immunotherapy. In preliminary experiments we could demonstrate that the combination of in vitro priming/in vivo boosting can yield significant titers of human antibodies against another human tumor associated antigen (ferritin). Furthermore, the feasibility of cloning human antibody genes from hu-SCID mice has recently been demonstrated (Nature (1992), 355:258). We believe that our novel combination approach can be extended to the generation of therapeutic antibodies against other diseases, beyond the currently targeted tumor associated antigens, and will become a general method for generating specific human MoAbs with high affinity.